January 2003
Volume 44, Issue 1
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Cornea  |   January 2003
Corneal Neovascularization after Excimer Keratectomy Wounds in Matrilysin-Deficient Mice
Author Affiliations
  • Tomoko Kure
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
  • Jin-Hong Chang
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
  • Takuji Kato
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
  • Everardo Hernández-Quintela
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
  • Hongqing Ye
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
  • Paul Chung-Shien Lu
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
  • Lynn M. Matrisian
    Department of Cell Biology, Vanderbilt University, Nashville, Tennessee;
  • Damien Gatinel
    Rothschild Foundation, Paris University, Paris, France; and the
  • Steven Shapiro
    Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts.
  • Faris Ghosheh
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
  • Dimitri T. Azar
    From the Massachusetts Eye and Ear Infirmary and
    Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the
Investigative Ophthalmology & Visual Science January 2003, Vol.44, 137-144. doi:https://doi.org/10.1167/iovs.01-1058
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      Tomoko Kure, Jin-Hong Chang, Takuji Kato, Everardo Hernández-Quintela, Hongqing Ye, Paul Chung-Shien Lu, Lynn M. Matrisian, Damien Gatinel, Steven Shapiro, Faris Ghosheh, Dimitri T. Azar; Corneal Neovascularization after Excimer Keratectomy Wounds in Matrilysin-Deficient Mice. Invest. Ophthalmol. Vis. Sci. 2003;44(1):137-144. https://doi.org/10.1167/iovs.01-1058.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Matrilysin, matrix metalloproteinase (MMP)-7, is upregulated in the corneal epithelium during wound healing after excimer keratectomy wounds. The purpose of this study was to determine the role of matrilysin in maintaining corneal avascularity during wound healing.

methods. Matrilysin-deficient mice (n = 17) and their age-matched wild-type littermates (n = 18) were treated with 193 nm argon-fluoride excimer keratectomy (experiment I). The percentage of corneal surface occupied by neovascularization was measured with a computer image-analysis program adjusted for parallax. In another experiment (experiment II), epithelial closure was monitored with slit lamp biomicroscopy and fluorescein staining, and corneal neovascularization was confirmed by india ink perfusion, electron microscopy, and immunolocalization of CD31 and type IV collagen. Corneal micropocket assays were performed to compare the area of corneal neovascularization in matrilysin-deficient mice and wild-type littermates (experiment III). To determine whether the differences in corneal neovascularization were related to differences in angiogenic factors, the levels of basic fibroblast growth factor (bFGF) were compared with those of vascular endothelial growth factor (VEGF) in matrilysin-deficient and wild-type mouse corneas (experiment IV).

results. The percentages of the corneal surface occupied by neovascularization after excimer laser keratectomy in the matrilysin-deficient mice measured 21.3% ± 5.2% and 18.7% ± 5.8% at days 3 and 7, respectively, compared with 5.3% ± 2.4% and 5.5% ± 3.4% in the wild-type littermates at days 3 (P < 0.01) and 7, respectively (P < 0.05; experiment I). No significant differences in the rates of epithelial closure of corneal wounds were observed between matrilysin-deficient and wild-type mice after wounding. Corneal neovascularization in the matrilysin-deficient mice was confirmed by india ink present in the corneal stromal blood vessels (extending from the limbus to the wound), immunohistochemical staining, and electron microscopy. Gram, Giemsa, calcofluor white, and acridine orange stains and electron microscopy showed no evidence of corneal infection (experiment II). The area of corneal neovascularization in matrilysin-deficient mice was not significantly different from that of wild-type littermates after implantation of bFGF pellets (0.91 ± 0.55 mm2 and 0.77 ± 0.34 mm2, respectively; experiment III). The levels of bFGF and VEGF (VEGF, VEGF-B, and VEGF-C) in corneal epithelial cells were not elevated in matrilysin-deficient mice compared with the wild-type mice (experiment IV).

conclusions. Matrilysin may play an important role in maintaining corneal avascularity during wound healing. The differences in corneal neovascularization between matrilysin-deficient mice and wild-type littermates seem unrelated to the bFGF and VEGF levels in the corneal epithelium.

Neovascularization is the formation of new vascular structures in areas that were previously avascular. Corneal neovascularization involves the sprouting of new vessels but does not ordinarily accompany corneal wound healing after excimer keratectomy. Several angiogenic factors, including basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-α and -β, have a vital role in corneal neovascularization. 1 In addition, several antiangiogenic factors, such as angiostatin, endostatin, and pigment epithelium-derived factor (PEDF), may play a role in the control of corneal neovascularization. 2 3 4  
Matrix metalloproteinases (MMPs) are proteolytic enzymes that contribute to degradation and remodeling of the extracellular matrix in both normal and pathologic conditions. 5 6 MMPs participate in many normal biological processes such as embryogenesis, morphogenesis, wound healing, and angiogenesis. More than 20 MMPs have been reported. They comprise several subgroups, including collagenases, gelatinases, stromelysins, membrane-bound MMPs, metalloelastase, and matrilysin. 5 6 7 8 9  
MMP-7 is the designated name of matrilysin. 9 Human MMP-7 complementary DNA was first isolated by Muller et al. 10 and rat and mouse cDNA sequences were determined later. 11 12 13 14 The zymogen of MMP-7 has a molecular mass of 28 kDa. When cleaved, the 19-kDa catalytic form is generated. MMP-7 cleaves matrix components such as fibronectin, gelatins (types I, III, IV, and V), collagen type IV, laminin, entactin-nidogen, and elastin. 15 16 17 18  
We have reported the expression of several MMPs, including MMP-7, in the cornea and their upregulation after corneal wounding. 19 20 These observations led us to investigate whether MMP-7 may contribute to the maintenance of corneal avascularity during wound healing. In this study, we assessed the involvement of MMP-7 in neovascularization after excimer keratectomy wounding in knockout and wild-type mouse models. 
Materials and Methods
Animals
The study was conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Animals ranged from 4 weeks to 4 months of age at the time of surgery. 
The matrilysin-deficient mice were generated as described by Wilson et al. 21 In MMP-7-knockout mice, a 6.5-kb BamHI genomic fragment cloned from a 129/Sv library was used to generate the targeting construct. A 550-bp EcoRV-StuI fragment spanning exons 3 and 4 was replaced with a 1.6-kb phosphoglycerate kinase-neomycin (PGK-neo) cassette. The construct was electroporated into R1 embryonic stem cells, and targeted clones were obtained and injected into C57BL/6 blastocysts after karyotype analysis. 
To generate wild-type littermates for MMP-7-knockout mice, MMP-7-knockout mice (experiments I and III) were crossed with wild-type mice (C57BL/6) to generate F1 heterozygotes (MMP-7+/−). MMP-7 heterozygotes were used to generate matrilysin-deficient mice and their wild-type littermates. Primers for wild-type alleles were located in exon 3 (5′-TCAGACTTACCTCGGATCGT-3′) and exon 4 (5′-GTCCTCACCATCAGTCCAG-3′), and primers for mutated alleles were located in the PGK-neo cassette (5′-TTGAGCCTGGCGAACAGT-3′ and 5′-TGGATTGCACGCAGGTTC-3′). 
Corneal Wound-Healing-Induced Neovascularization: Experiment I
Matrilysin-deficient and C57BL/6 littermate mice, each weighing 15 to 25 g, were included in the study. The C57BL/6 mice served as the control. Anesthesia was administered by intramuscular injection of a mixture of ketamine (200 mg/kg) and xylazine (10 mg/kg). Topical anesthesia (proparacaine 0.5%) was also used. After epithelial debridement with a number 15 Beaver blade, 75 to 100 pulses with a 1.8-mm diameter excimer keratectomy (193-nm argon-fluoride laser with fluence set at 160 mJ/cm2 and repetition rate of 5 Hz) were applied to matrilysin-deficient (n = 17) and littermate (n = 18) mice. Topical erythromycin ointment (0.3%) was applied immediately after surgery. The eyes were examined on postoperative days 3 and 7 by slit lamp microscopy and photographed at standardized magnification using a slit lamp camera (Nikon, Melville, NY). 
The percentage of corneal surface occupied by neovascularization was calculated on computer (NIH Image; Scion, Frederick, MD), as described by Moromizato et al., 22 with modifications. Briefly, the photographs of mouse corneas were digitized and the images were fed into a computer (Vaio, model PCG-F370; Sony, Tokyo, Japan) and converted to tagged information file format (TIFF). The images were resolved and converted to NIH Image. The total corneal area and the area of neovascularization were outlined. To minimize the parallax problem expected to distort the actual size of the area of neovascularization, the ratios of neovascular cornea over total corneal area were measured from the same image. The angle of photographic documentation was estimated to within ±5°, and the measurements of percentage of neovascularization was corrected using the parallax tables derived from simulations of various angles of observations (ranging from 5° to 90° in 5° increments) and of various percentages of neovascularization (ranging from 0%–100% in 10% increments). Boolean operations performed on computer (Bryce 3-D; Metacreations, Dublin, Ireland) were used to model three dimensional (3-D) concentric corneal neovascularization of a mouse cornea. Two-dimensional (2-D) images were generated for each angle of observation and percentage of neovascularization (slit lamp simulations). The computer was used (NIH Image; Scion) to measure apparent percentage of neovascularization of the 2-D simulation and to estimate the short- and long-axis ratios of the images, which allowed the generation of our parallax correction tables. 
Corneal Reepithelialization Rates and India Ink Perfusion: Experiment II
Additional matrilysin-deficient (n = 23) and wild-type (n = 27) mice were generated by crossing homozygous mice. These mice were treated with laser keratectomy, as described earlier. Clinical slit lamp examination and fluorescein staining were performed 6, 18, and 24 hours and 2, 3, 4, 5, and 7 days after excimer keratectomy. Digitized scans of slit lamp photographs with fluorescein staining were used to calculate the apparent rate of corneal reepithelialization (PhotoShop ver 4.0.1; Adobe, San Jose, CA). Graphic analysis of linear regression was used to determine the apparent rate of resurfacing after wounding. 
The angle of photographic documentation was also estimated to within ±5° and measurements of the corrected percentage of epithelialization were performed using the parallax tables, as described herein. 
In addition to slit lamp photography, india ink was used to confirm the presence of neovascularization. Mice were killed with a lethal dose of intraperitoneal pentobarbital sodium. After perfusion of the bodies with 50 mL lactated Ringer’s solution to completely remove blood from the vessels, 20 mL 10% india ink (Eberhard Faber, Lewisburg, TN) in Ringer’s solution was added immediately, followed by 20 mL of gelatin mixture (6% gelatin; Sigma, St. Louis, MO) and 6% india ink in Ringer’s solution filtered through a paper filter (No. 41 filter; Whatman International, Maidstone, UK). The gelatin mixture within the corneal vessels was solidified by freezing at −20°C for 5 minutes. The eyes were enucleated and fixed in 10% formaldehyde (PBS neutralized) for 24 hours. The cornea and 1-mm rim of adjacent scleral tissue were separated from the globe, and three full-thickness peripheral radial cuts were made through the cornea to allow flattening. The corneas were placed on slides and photographed. 
Histologic Examination, Immunohistochemical Staining, Confocal Microscopy and Electron Microscopy: Experiment II
Used for immunohistochemistry were goat anti-human/bovine type IV collagen polyclonal antibody (Southern Biotechnology, Birmingham, AL) and rat anti-mouse CD31 monoclonal antibody (PharMingen, San Diego, CA). Type IV collagen is the major collagenous component of blood vessel basement membranes, and its distribution in ocular tissue was examined. 23 24 25 CD31 is expressed on the surface of endothelial cells. 26 We used the antibodies for detecting blood vessels in the cornea. Rhodamine red-X-conjugated affinity-purified anti-goat IgG antibody and fluorescein-conjugated affinity-purified anti-rat IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) were used as secondary antibodies. 
The eyes were embedded in optimal cutting temperature (OCT) compound (Miles, Elkhart, IN), frozen in liquid nitrogen, and stored at −80°C until sectioning. Frozen 6- to 8-μm-thick sections were cut with a cryostat and mounted on slides (Fisher Scientific, Pittsburgh, PA). Histologic examination was performed after hematoxylin and eosin, Gram, Giemsa, calcofluor white, and acridine orange staining. 
The tissue sections were incubated with 1% bovine albumin fraction V at room temperature for 30 minutes and then incubated with primary antibodies (1:100 dilution) at room temperature for 1 hour. The slides were washed with PBS three times for 5 minutes and incubated with secondary antibodies (1:400 dilution) at room temperature for 30 minutes. The slides were washed in PBS, mounted with antifade medium (Vectashield; Vector, Burlingame, CA), and examined by laser scanning confocal microscopy (Leica, Deerfield, IL). Negative control was prepared in the same manner, with 1% bovine albumin fraction V used only for the primary antibody incubation step. 
For electron microscopy, eyes were fixed in half-strength Karnovsky fixative (2% paraformaldehyde and 2.5% glutaraldehyde) in 0.2 M sodium cacodylate buffer (pH 7.4) overnight and postfixed in 1% osmium tetroxide in 0.2 M sodium cacodylate for 1.5 hours. After dehydration in graded alcohol, the eyes were embedded in Epon. Ultrathin sections (8–9 nm) stained with 2% uranyl acetate and Reynold’s lead nitrate were studied by electron microscopy. 
Corneal Micropocket Assay: Experiment III
The mouse corneal micropocket assay was performed according to the procedure described by Kenyon et al. 27 Briefly, mice were anesthetized by ketamine and xylazine injection. Proparacaine was used for local anesthesia. Corneal micropockets were created with a modified von Graefe knife in MMP-7-deficient mice (n = 7) and wild-type littermates (n = 7). Hydron pellets (0.4 × 0.4 mm) containing 30 or 40 ng bFGF and 40 μg sucrose aluminum sulfate were implanted into the corneal pockets. Ofloxacin eye drops were applied after surgery. The eyes were examined by slit lamp microscopy 3 and 7 days after surgery. The neovascularization area (A) was estimated based on the length and clock hours of corneal vessel involvement, as described previously, with modification 27
A ≅ 0.5πR 1 R 2, where R 1 is maximum length of the vessels (in millimeters) and R 2 is π/6 × clock hours of neovascularization (calculated from the perimeter of a mouse cornea with a 2-mm radius). 
Western Blot Analysis: Experiment IV
Mouse corneal epithelial cells (wild-type and matrilysin-deficient mice) were collected by scraping. The cells were lysed by lysis buffer and run on 10% to 20% precast SDS polyacrylamide gels (Novex, San Diego, CA) and then electrotransferred onto nylon membranes (Immobilon P; Millipore, Bedford, MA). Membranes were blotted with anti-VEGF (Calbiochem, San Diego, CA), anti-bFGF (Oncogene, Cambridge, MA), anti-VEGF-B (R&D Systems, Minneapolis, MN), and anti-VEGF-C (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies and reacted with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG secondary antibodies (NEN Life Science Products, Boston, MA). After three washes with Tris-buffered saline-Tween-20 (TBST) for 15 minutes, immunoblots were developed with enhanced chemiluminescence (ECL) reagent (Western blot detection reagents; NEN Life Science Products). 
Statistical Analysis
Statistical analysis was performed on computer with Student’s non-paired t-test and analysis of variance (Statview, ver. 5.0; SAS Institute Inc., Cary, NC). P < 0.05 was deemed significant. 
Results
Corneal Wound Healing-Induced Neovascularization: Experiment I
PCR was used to confirm the genotyping of matrilysin-deficient and wild-type animals (Fig. 1A) . Not taking into consideration the parallax problem, we calculated the apparent percentage of corneal surface occupied by neovascularization in matrilysin-deficient mice at days 3 and 7 to be 26.6% ± 5.5% and 23.4% ± 6.6%, respectively, which were significantly greater than in wild-type littermates (6.4% ± 2.7% and 6.6% ± 3.9%, days 3 and 7; P < 0.05 at days 3 and 7, respectively). 
When we adjusted the percentages of corneal neovascularization for parallax, the percentages of the corneal surface occupied by neovascularization in matrilysin-deficient mice at day 3 and 7 were 21.3% ± 5.23% and 18.7% ± 5.8%, respectively, significantly greater than in wild-type littermates (5.33% ± 2.39% and 5.5% ± 3.36%, day 3 and day 7, respectively); day 3: P < 0.05, day 7: P < 0.05; Fig. 1B ). 
The number of mice having no, 0% to 10%, 10.1% to 20%, and more than 20% neovascularization was computed for days 3 and 7. The ratio of mice with more than 10% estimated neovascularization was greater in the MMP-7-deficient mice (9/17 and 6/17 at days 3 and 7, respectively) compared with the wild-type littermates (2/18; Fig. 1C ). 
Corneal Reepithelialization Rates: Experiment II
All wounded corneas were completely reepithelialized within 2 to 5 days (Fig. 2) . Not taking into consideration the parallax problem, we calculated the apparent epithelial closure rates of the wounds to be 7.6 × 10−2 mm2/h and 7.0 × 10−2 mm2/h in matrilysin-deficient and wild-type mice, respectively. The differences between the groups were not statistically significant. 
We adjusted the percentage of epithelialization at 6, 18, 24, and 48 hours for parallax (Fig. 2) , and observed that the percentages of epithelial closure at 6, 18, 24, and 48 hours were 78 8% ± 4.8%, 59% ± 7.4%, 45.8% ± 13.7%, and 1% ± 2.2% for wild-type mice, respectively. The percentage of epithelial closure at 6, 18, 24, and 48 hours were 71.8% ± 8%, 52.4% ± 10%, 39.8% ± 3.9% and 0% ± 0% in matrilysin-deficient mice, respectively. The differences between the two groups were not statistically significant (P = 0.3). 
Corneal Neovascularization in Matrilysin-Deficient Mice: Experiment II
Substantial neovascularization after corneal wounding was observed at 1 week in 3.7% (1/27) of corneas of wild-type mice and 30.4% (7/23) of corneas of MMP-7-knockout mice. The presence of vessels in the corneas was confirmed by perfusion with india ink and by electron microscopic examination (Fig. 3) . Electron microscopic examination and Gram, Giemsa, calcofluor white, and acridine orange stains showed evidence of blood vessels and erythrocytes in the corneal stroma, but no evidence of bacterial, mycotic, or parasitic corneal infection. Immunohistochemical staining showed evidence of colocalization of anti-type IV collagen and anti-CD31 antibodies in the lumen of stromal vessels. 
FGF Response in Matrilysin-Deficient Mice: Experiment III
To determine whether corneal vascularization was altered in matrilysin-deficient mice, micropellets of the slow-release polymer hydron containing bFGF were implanted into the corneas of matrilysin-deficient mice and their wild-type littermates. By day 7, all wild-type (Fig. 4A) and matrilysin-deficient (Fig. 4B) mice had corneal neovascularization extending more than 0.1 mm from the limbus, visible by slit lamp examination. The corneas were photographed on days 3, 5, and 7 after implantation and total area of neovascularization was estimated. The induced neovascular areas were 0.23 ± 0.3, 0.47 ± 0. 16, and 0.77 ± 0.34 mm2 in the wild-type littermates and 0.30 ± 0.26, 0.44 ± 0.13, and 0.91 ± 0.55 mm2 in the matrilysin-deficient mice at days 3, 5, and 7, respectively. The differences between the two groups were not statistically significant (Fig. 4C)
Western Blot Analysis of bFGF and VEGF in Wild-type and Matrilysin-Deficient Littermates: Experiment IV
To determine whether the levels of angiogenic factors are elevated in MMP-7-knockout mice, corneas of wild-type and MMP-7-deficient mice were scraped (n = 4 per group). Corneal epithelium was subjected to Western blot analysis with anti-bFGF, VEGF, VEGF-B, and VEGF-C antibodies. The levels of bFGF (Fig. 4D) and the VEGFs (Figs. 4E 4F 4G) in wild-type corneas (lane 1) were higher than those in MMP-7-knockout mice (lane 2). Another scraping of resurfaced epithelium at day 6 showed that corneal epithelial bFGF and VEGF levels were not elevated after wounding. 
Discussion
Our findings suggest a role for MMP-7 in maintaining corneal clarity after keratectomy wounding. A substantially higher level of corneal neovascularization developed in MMP-7-knockout mice after excimer keratectomy wounding than did age-matched wild-type mice littermates. The presence of vessels was confirmed by india ink perfusion, electron microscopy, and immunohistochemical localization of type IV collagen and CD31. There was no increase in corneal reepithelialization rates, response to implanted bFGF pellets, and levels of bFGF, VEGF, VEGF-B, and VEGF-C in the matrilysin-deficient compared with the wild-type mice. 
Corneal avascularity is essential for corneal transparency and optimal visual performance. Most research on corneal vascularization has focused on the upregulation of angiogenic factors in diseased corneas, reflecting current views of tumor-related angiogenesis. Some reports suggest that the induction of new vessels involves not only the activation of angiogenic factors such as vascular endothelial growth factor and fibroblast growth factor, 22 28 but also the suppression of antiangiogenic factors. Angiogenic factors, such as bFGF, have been shown to play a role in the regulation of corneal angiogenesis in a dose- and species-dependent manner. Seghezzi et al. 29 noted increased levels of VEGF in vascular endothelial cells and demonstrated that a neutralizing antibody to VEGF decreases bFGF-induced corneal neovascularization. In this study, we also found that bFGF induced corneal neovascularization. These findings suggest that growth factors have a strong impact on corneal neovascularization. In MMP-7-knockout mice, we did not find elevated levels of bFGF and VEGF in the corneal epithelial cells. This suggests that factors other than bFGF and VEGF may be involved in corneal neovascularization or that these factors may be elevated in the stromal or endothelial layers of the cornea. 
Matrilysin is constitutively expressed in epithelial cells. We have shown that matrilysin is expressed at the leading edge and in the basal epithelial cells in a rat model of wound healing. 19 Di Girolamo et al. 30 have also demonstrated positive matrilysin staining in the basal epithelial cells of pterygium specimens. They concluded that MMP-7 may play a role in the migration and proliferation phase of wound healing, implicating its potential function in the pathogenesis of the disease and angiogenesis in pterygium. In addition, matrilysin may have a role in maintaining corneal avascularity. It has been shown that MMP-7 cleaves decorin, an extracellular matrix protein, which may regulate and release TGF-β1, which is known to be antiangiogenic in the extracellular matrix. 31 Recently, we have documented that MMP-7 cleaves corneal collagen XVIII to generate a 28-kDa fragment. 32 This MMP-7-generated fragment contains the endostatin domain of collagen XVIII which has a potent antiangiogenic function. 
Various mechanisms may explain the effect of MMP-7 on vessel formation. Vascular vessel formations are regulated by the balance of antiangiogenic factors and angiogenic factors. Several angiogenic factors such as VEGF and bFGF, bind to their cognate receptor and enhance vascular endothelial cell proliferation. In contrast, antiangiogenic factors, such as angiostatin and endostatin, are derived from proteolytic cleavage of their precursor molecules and generate functional fragments. Angiostatin and endostatin have been shown to be effective in blocking vascular endothelial cell proliferation and may contribute to regression in the cornea. The fact that MMP-7 cleaves plasminogen and collagen XVIII in vitro to generate angiostatin and endostatin, respectively, leads us to hypothesize that the reduction of MMP-7-derived angiostatin and/or endostatin in the cornea may contribute to corneal neovascularization after excimer laser keratectomy in matrilysin-knockout animals. It is also possible that MMP-7 has a direct effect on vessel formation in the cornea. Nishizuka et al. 33 have shown that MMP-7 stimulates DNA synthesis of cultured umbilical vein endothelial cells and induces angiogenesis in vivo. They also note that subcutaneous injections of antisense oligonucleotides reduces blood vessel formation. 
We have used two methods to estimate the extent of corneal neovascularization for the pellet experiments. We used the approximations of Kenyon et al., 27 with modification (experiment III), and, for the concentric neovascularization and wound healing (experiments I and II), we used a computer-generated model to compare the measured and actual angles and areas of neovascularization in mouse corneas (Fig. 5) . This model takes the curvature of the mouse cornea into consideration and allows us to determine the difference between measured and actual corneal neovascularization through slit lamp photography. Although this model provides more accurate calculations of the percentage of corneal neovascularization, we could not use it for the measurement of vessel density and corneal pocket pellet (bFGF)-induced corneal neovascularization. (Pellet-induced corneal neovascularization is sectoral; it does not develop evenly and is not concentric with the limbus [experiment III].) The half-ellipse method of calculation used 27 is only an approximation, which may have contributed to the absence of significant differences in neovascularization after pellet surgery. 
An interesting finding in the present study is the diminished expression of bFGF and VEGF in the corneal epithelium of the matrilysin-knockout compared with the wild-type mouse. One explanation is that knocking out the MMP-7 gene may have diminished the expression of VEGF, VEGF-B, VEGF-C, and bFGF. However, it is unlikely that the reduced levels of these four factors in the corneal epithelium contributed to the increased corneal neovascularization in the matrilysin-knockout animals. Our data presented in Figure 4 , suggest that the contribution of VEGF, VEGF-B, VEGF-C, and bFGF by the corneal epithelium does not explain the major finding of this study—namely, the increased corneal vascularization in MMP-7-knockout compared with wild-type mice. 
In summary, we have reported the involvement of MMPs in corneal wound healing after laser keratectomy in prior reports. 19 20 34 35 Enhanced corneal neovascularization occurs after keratectomy wounds in MMP-7-knockout mice, suggesting that MMP-7 may play a role in maintaining corneal avascularity during wound healing. The findings of the present study may provide evidence of a correlation between MMPs and corneal neovascularization. However, it is not clear whether the primary inhibitory effect of MMP-7 on formation of blood vessels is a direct effect or is secondary to its proteolytic cleavage action to generate intermediate functional antiangiogenic fragments. Further investigations of angiogenic and antiangiogenic factors generated by MMP-7 processing will improve our understanding of the role of MMP-7 in maintaining corneal avascularity. 
 
Figure 1.
 
Enhanced corneal neovascularization in matrilysin-deficient mice compared with that of wild-type littermates. (A) Tail genomic DNAs for matrilysin-deficient mice were amplified by PCR. The PCR fragments representing normal alleles are 700 bp and mutant alleles are 500 bp for MMP-7-knockout mice (+/+, wild-type; +/−, heterozygous; −/−, homozygous). (B) Mice were treated with the excimer laser and the percentages of the corneal surface occupied by vascularization were determined at days 3, and 7 by digitized analysis of slit lamp photography. *Statistically significant differences (P < 0.05). (C) Percentages of corneal neovascularization in MMP-7-knockout mice and wild-type littermates were determined at days 3 and 7 after corneal wounding. Data are number of animals in each category.
Figure 1.
 
Enhanced corneal neovascularization in matrilysin-deficient mice compared with that of wild-type littermates. (A) Tail genomic DNAs for matrilysin-deficient mice were amplified by PCR. The PCR fragments representing normal alleles are 700 bp and mutant alleles are 500 bp for MMP-7-knockout mice (+/+, wild-type; +/−, heterozygous; −/−, homozygous). (B) Mice were treated with the excimer laser and the percentages of the corneal surface occupied by vascularization were determined at days 3, and 7 by digitized analysis of slit lamp photography. *Statistically significant differences (P < 0.05). (C) Percentages of corneal neovascularization in MMP-7-knockout mice and wild-type littermates were determined at days 3 and 7 after corneal wounding. Data are number of animals in each category.
Figure 2.
 
Time course of corneal reepithelialization (A) Corneal reepithelialization after corneal wounding. (Aa) Wild-type mouse; (Ab) MMP-7 knockout mouse. Fluorescein was applied over the cornea before slit lamp photography. (B) Graphic representation of the kinetics of corneal reepithelialization after corneal wounding. The residual epithelial defect was evaluated by fluorescein staining at 6, 18, 24, and 48 hours after corneal wounding. The size of the remaining epithelial defect was established for each mouse, and the mean values were plotted. Bars indicate SD.
Figure 2.
 
Time course of corneal reepithelialization (A) Corneal reepithelialization after corneal wounding. (Aa) Wild-type mouse; (Ab) MMP-7 knockout mouse. Fluorescein was applied over the cornea before slit lamp photography. (B) Graphic representation of the kinetics of corneal reepithelialization after corneal wounding. The residual epithelial defect was evaluated by fluorescein staining at 6, 18, 24, and 48 hours after corneal wounding. The size of the remaining epithelial defect was established for each mouse, and the mean values were plotted. Bars indicate SD.
Figure 3.
 
Corneal neovascularization in MMP-7 knockout mouse after keratectomy wound. Photographs show (A) clear cornea before wounding and (B) corneal neovascularization 2 weeks after wounding, with many new vessels extending from the limbus. (C) Appearance of vessels in the cornea after india ink perfusion. (D) Enlarged view of perfused vessels. (E) Electron microscopy of cornea exhibiting neovascularization after keratectomy wounding. Blood vessels were present in the stroma (✶), with calcium deposition between epithelium and stroma (arrows). (FI) Histologic examination and immuno- histochemical staining of corneas exhibiting neovascularization after keratectomy wounding. Clinical appearance of corneal neovascularization persisted for 2 months after wounding. Immunohistochemical staining using confocal microscopy of frozen sections with antibodies against type IV collagen (F) against vascular endothelial cells (CD31; G) and negative control (I). (H) Double staining illustrates colocalization of type IV collagen and vascular endothelial cells in the corneal stroma. Scale bars: (C) 1 μm; (D) 100 μm; (E) 10 μm; (FI) 50 μm.
Figure 3.
 
Corneal neovascularization in MMP-7 knockout mouse after keratectomy wound. Photographs show (A) clear cornea before wounding and (B) corneal neovascularization 2 weeks after wounding, with many new vessels extending from the limbus. (C) Appearance of vessels in the cornea after india ink perfusion. (D) Enlarged view of perfused vessels. (E) Electron microscopy of cornea exhibiting neovascularization after keratectomy wounding. Blood vessels were present in the stroma (✶), with calcium deposition between epithelium and stroma (arrows). (FI) Histologic examination and immuno- histochemical staining of corneas exhibiting neovascularization after keratectomy wounding. Clinical appearance of corneal neovascularization persisted for 2 months after wounding. Immunohistochemical staining using confocal microscopy of frozen sections with antibodies against type IV collagen (F) against vascular endothelial cells (CD31; G) and negative control (I). (H) Double staining illustrates colocalization of type IV collagen and vascular endothelial cells in the corneal stroma. Scale bars: (C) 1 μm; (D) 100 μm; (E) 10 μm; (FI) 50 μm.
Figure 4.
 
Corneal neovascularization after bFGF pellet implantation. Corneal neovascularization was seen in (A) wild-type and (B) matrilysin-deficient mice at day 7. (C) Corneal neovascularization was quantified on days 3, 5, and 7 after pellet implantation. For Western blot analysis of bFGF, VEGF, VEGF-B, and VEGF-C, corneal epithelial cells of wild-type and MMP-7-knockout mice were scraped and lysed with lysis buffer, run on SDS-polyacrylamide gels, and subjected to Western blot analysis with anti-bFGF antibody (D), anti-VEGF antibody (E), anti-VEGF-B antibody (F), and anti-VEGF-C antibody (G). Corneal epithelial cell lysates of (lane 1) wild-type mice and (lane 2) matrilysin-deficient mice.
Figure 4.
 
Corneal neovascularization after bFGF pellet implantation. Corneal neovascularization was seen in (A) wild-type and (B) matrilysin-deficient mice at day 7. (C) Corneal neovascularization was quantified on days 3, 5, and 7 after pellet implantation. For Western blot analysis of bFGF, VEGF, VEGF-B, and VEGF-C, corneal epithelial cells of wild-type and MMP-7-knockout mice were scraped and lysed with lysis buffer, run on SDS-polyacrylamide gels, and subjected to Western blot analysis with anti-bFGF antibody (D), anti-VEGF antibody (E), anti-VEGF-B antibody (F), and anti-VEGF-C antibody (G). Corneal epithelial cell lysates of (lane 1) wild-type mice and (lane 2) matrilysin-deficient mice.
Figure 5.
 
Model for setting actual percentage of corneal neovascularization: a 3-D computer-generated model of concentric corneal neovascularization and 10% corneal neovascularization. This model was used to calculate the surface area of NV. The surface area of a spherical zone was calculated by S = 2πRh, where R is the radius of the sphere and h is height of the zone.
Figure 5.
 
Model for setting actual percentage of corneal neovascularization: a 3-D computer-generated model of concentric corneal neovascularization and 10% corneal neovascularization. This model was used to calculate the surface area of NV. The surface area of a spherical zone was calculated by S = 2πRh, where R is the radius of the sphere and h is height of the zone.
The authors thank Melodie Henderson for assistance with mouse husbandry. 
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Figure 1.
 
Enhanced corneal neovascularization in matrilysin-deficient mice compared with that of wild-type littermates. (A) Tail genomic DNAs for matrilysin-deficient mice were amplified by PCR. The PCR fragments representing normal alleles are 700 bp and mutant alleles are 500 bp for MMP-7-knockout mice (+/+, wild-type; +/−, heterozygous; −/−, homozygous). (B) Mice were treated with the excimer laser and the percentages of the corneal surface occupied by vascularization were determined at days 3, and 7 by digitized analysis of slit lamp photography. *Statistically significant differences (P < 0.05). (C) Percentages of corneal neovascularization in MMP-7-knockout mice and wild-type littermates were determined at days 3 and 7 after corneal wounding. Data are number of animals in each category.
Figure 1.
 
Enhanced corneal neovascularization in matrilysin-deficient mice compared with that of wild-type littermates. (A) Tail genomic DNAs for matrilysin-deficient mice were amplified by PCR. The PCR fragments representing normal alleles are 700 bp and mutant alleles are 500 bp for MMP-7-knockout mice (+/+, wild-type; +/−, heterozygous; −/−, homozygous). (B) Mice were treated with the excimer laser and the percentages of the corneal surface occupied by vascularization were determined at days 3, and 7 by digitized analysis of slit lamp photography. *Statistically significant differences (P < 0.05). (C) Percentages of corneal neovascularization in MMP-7-knockout mice and wild-type littermates were determined at days 3 and 7 after corneal wounding. Data are number of animals in each category.
Figure 2.
 
Time course of corneal reepithelialization (A) Corneal reepithelialization after corneal wounding. (Aa) Wild-type mouse; (Ab) MMP-7 knockout mouse. Fluorescein was applied over the cornea before slit lamp photography. (B) Graphic representation of the kinetics of corneal reepithelialization after corneal wounding. The residual epithelial defect was evaluated by fluorescein staining at 6, 18, 24, and 48 hours after corneal wounding. The size of the remaining epithelial defect was established for each mouse, and the mean values were plotted. Bars indicate SD.
Figure 2.
 
Time course of corneal reepithelialization (A) Corneal reepithelialization after corneal wounding. (Aa) Wild-type mouse; (Ab) MMP-7 knockout mouse. Fluorescein was applied over the cornea before slit lamp photography. (B) Graphic representation of the kinetics of corneal reepithelialization after corneal wounding. The residual epithelial defect was evaluated by fluorescein staining at 6, 18, 24, and 48 hours after corneal wounding. The size of the remaining epithelial defect was established for each mouse, and the mean values were plotted. Bars indicate SD.
Figure 3.
 
Corneal neovascularization in MMP-7 knockout mouse after keratectomy wound. Photographs show (A) clear cornea before wounding and (B) corneal neovascularization 2 weeks after wounding, with many new vessels extending from the limbus. (C) Appearance of vessels in the cornea after india ink perfusion. (D) Enlarged view of perfused vessels. (E) Electron microscopy of cornea exhibiting neovascularization after keratectomy wounding. Blood vessels were present in the stroma (✶), with calcium deposition between epithelium and stroma (arrows). (FI) Histologic examination and immuno- histochemical staining of corneas exhibiting neovascularization after keratectomy wounding. Clinical appearance of corneal neovascularization persisted for 2 months after wounding. Immunohistochemical staining using confocal microscopy of frozen sections with antibodies against type IV collagen (F) against vascular endothelial cells (CD31; G) and negative control (I). (H) Double staining illustrates colocalization of type IV collagen and vascular endothelial cells in the corneal stroma. Scale bars: (C) 1 μm; (D) 100 μm; (E) 10 μm; (FI) 50 μm.
Figure 3.
 
Corneal neovascularization in MMP-7 knockout mouse after keratectomy wound. Photographs show (A) clear cornea before wounding and (B) corneal neovascularization 2 weeks after wounding, with many new vessels extending from the limbus. (C) Appearance of vessels in the cornea after india ink perfusion. (D) Enlarged view of perfused vessels. (E) Electron microscopy of cornea exhibiting neovascularization after keratectomy wounding. Blood vessels were present in the stroma (✶), with calcium deposition between epithelium and stroma (arrows). (FI) Histologic examination and immuno- histochemical staining of corneas exhibiting neovascularization after keratectomy wounding. Clinical appearance of corneal neovascularization persisted for 2 months after wounding. Immunohistochemical staining using confocal microscopy of frozen sections with antibodies against type IV collagen (F) against vascular endothelial cells (CD31; G) and negative control (I). (H) Double staining illustrates colocalization of type IV collagen and vascular endothelial cells in the corneal stroma. Scale bars: (C) 1 μm; (D) 100 μm; (E) 10 μm; (FI) 50 μm.
Figure 4.
 
Corneal neovascularization after bFGF pellet implantation. Corneal neovascularization was seen in (A) wild-type and (B) matrilysin-deficient mice at day 7. (C) Corneal neovascularization was quantified on days 3, 5, and 7 after pellet implantation. For Western blot analysis of bFGF, VEGF, VEGF-B, and VEGF-C, corneal epithelial cells of wild-type and MMP-7-knockout mice were scraped and lysed with lysis buffer, run on SDS-polyacrylamide gels, and subjected to Western blot analysis with anti-bFGF antibody (D), anti-VEGF antibody (E), anti-VEGF-B antibody (F), and anti-VEGF-C antibody (G). Corneal epithelial cell lysates of (lane 1) wild-type mice and (lane 2) matrilysin-deficient mice.
Figure 4.
 
Corneal neovascularization after bFGF pellet implantation. Corneal neovascularization was seen in (A) wild-type and (B) matrilysin-deficient mice at day 7. (C) Corneal neovascularization was quantified on days 3, 5, and 7 after pellet implantation. For Western blot analysis of bFGF, VEGF, VEGF-B, and VEGF-C, corneal epithelial cells of wild-type and MMP-7-knockout mice were scraped and lysed with lysis buffer, run on SDS-polyacrylamide gels, and subjected to Western blot analysis with anti-bFGF antibody (D), anti-VEGF antibody (E), anti-VEGF-B antibody (F), and anti-VEGF-C antibody (G). Corneal epithelial cell lysates of (lane 1) wild-type mice and (lane 2) matrilysin-deficient mice.
Figure 5.
 
Model for setting actual percentage of corneal neovascularization: a 3-D computer-generated model of concentric corneal neovascularization and 10% corneal neovascularization. This model was used to calculate the surface area of NV. The surface area of a spherical zone was calculated by S = 2πRh, where R is the radius of the sphere and h is height of the zone.
Figure 5.
 
Model for setting actual percentage of corneal neovascularization: a 3-D computer-generated model of concentric corneal neovascularization and 10% corneal neovascularization. This model was used to calculate the surface area of NV. The surface area of a spherical zone was calculated by S = 2πRh, where R is the radius of the sphere and h is height of the zone.
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